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PostPosted: Mon Jun 04, 2007 5:25 pm 
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Joined: Sun May 30, 2004 11:15 am
Posts: 198
Location: Canada
Thanks for the very valuable tips. Like TxRob, I don't keep sterilized water on hand and I don't have confidence that it stays sterilized for very long. I like the reassurance that 3% bleach gives me.

This last batch of stem, I recut the bottom of the node in an effort to provide better contact with the media. All the times befroe that I have done nodes in a glove box and didn't have the room to recut. I had good or better success without doing a fresh cut after sterilizing.

What is everyones thoughts on cutting the stems before instering them into the media?

Thanks!

Kyle


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PostPosted: Mon Jun 04, 2007 8:33 pm 
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Joined: Sun Dec 14, 2003 6:06 pm
Posts: 2173
Location: Kanab, Utah
Success with stems is relative. I believe that you can have "success" with doing a lot of things that, for one reason or another, I no longer do with stems. It is entirely possible to fine tune seveal different aspects of doing stems for increased "success". I measure success in doing stems by the rate of growth, number of nodes that grow to plants and number of plants per node. I have recently downplayed the number of plants per node because I see a possiblity for mutation when one ups the amount of BAP, especially in the presence of coconut water.
It is possible to grow good stems at an "acceptable" percent success rate without cutting the stems after bleach treatment. It is also possible to grow a significant rate of plants per node with a 3% final rinse in bleach. It is also possible to improve on all of these treatment protocols. Each person doing this type of work has to decide at what level do I call this saitisfactory for my purposes.
I always cut the stems before inserting them in the medium despite the fact that I had a series of tests that indicated that this increased phenol production.
A note about sterile water. For many purposes, I use 20 ml ampules with a membrane that I obtain from my local pharmacy. These have a very long shelf life and are small enough that once you open the top and expose the membrane you will probably use it up before anything bad can happen. I batch sterilize larger amounts of water in the pressure cooker for other puposes but I always use the pharmacy sterile water ampules for anything that I can't afford to lose, like seed.
Dean

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PostPosted: Tue Jun 05, 2007 8:59 am 
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Joined: Tue Jun 01, 2004 11:43 pm
Posts: 1027
Location: Canyon Country, Ca
Dean, I think we all agree using the sterile water rinse would be better. I can see if you are purchasing it from the pharmacy that there would be an added level of confidence that what you are using is really sterile. Beyond that it’s really about having appropriate storage vessels on hand that you can autoclave and ensure they stay sterile after the fact.

Personally I haven't been doing stem props for much longer than a couple of years. Back when I first started I had a horrible time with contamination. So I had to really work on my process to get the kinks out and started using the heavier protocol to clean up my stems. The changes I made worked and significantly increased my success rate from about 10% to about 80-90% even with older stems. Now that I have the basic techniques working, I can spend more time focusing on optimizing what I’m doing.

Originally I used to do a “sterile” water rinse back when I had a horrible success rate. But I was concerned that might have been one of the avenues for potential contamination if I was accidentally contaminating my water at some stage in the process. So I went back to the basic protocol to try to eliminate any errors on my part.

Now that my current process has been working for a while and I’m in the grove I can start introducing one specific change at time into my protocol. That way I’ll be able to tell if it’s a safe change based on my results. (vs. worrying about multiple potential issues and not knowing which one is the problem.)

So I completely see why people would want to start with the 3% final stage until they have their basic technique down. Stems are not the easiest of things to get going. It definitely takes practice to get them sterile without frying them and then keeping them sterile until you get them into tubes.

Beyond that though, any experienced individual doing this type of work should be constantly looking for ways to improve their technique for better results. So its obviously very valid to change to this step.

:-)

Rob


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PostPosted: Tue Jun 05, 2007 9:12 am 
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Joined: Thu Jan 05, 2006 1:28 pm
Posts: 1826
Location: Santa Cruz CA USA
I cook my own sterile distilled water in 500 ml narrow neck Erlenmeyers with typical one hole stoppers with cotton wadding. I only put about 400 ml of water in them, as that is about how much I tend to use at once. I cover the tops with Al foil. I keep them in a cool dark place after sterilizing with the pressure cookers. I use this water months later, I have never had a problem. I use the most sterile water when I am sowing seed; I rarely contaminate seed, even green cap.

Once I open a flask of sterile water in the hood, I only use it for that session; I do not try to reuse it later without re-sterillizing it.

This a cheap and easy way to have sterile distilled water on hand.

As far as phenolics go, I do not have what I perceive to be a problem. I rarely have to transflask stems due to phenolics. I do keep track of why I transflask stems in a database, and phenolic production is one of the reasons. However over time that number has dropped drastically. The single biggest change was when I stopped "scraping off the bract" using forceps. The amount of tissue damage that caused, did create a lot of phenols. When I went to surgically removing the bract with a scalpel, the number of transflaskings due to phenols went way down. Do I think I would probably have "better" results by rinsing at the end with sterile distilled water? Probably. I also think I would probably occasionally have to take stems out and re-sterilize them, which I rarely have to do now. ;-)

As Dean pointed out, there are many ways to do this, and many metrics to use to determine if you are "successful". You can sometimes get keikis on stems that you simply place in water or on moss; or you can spend a great deal of time (years) tuning your process so that it not only reliably provides the number of plants that you want, but it also works with your available skill level and resources. The first stems I did on plain Knudsen C twenty years ago survived in spite of every mistake I could make. ;-) I now do a significantly better job. I imagine I will continue to improve my process, because I am never satisfied. That is one great reason for participating in fora like this one: you can learn a lot by listening to what other people have success with, and adapting your own process to mitigate its shortcomings. "Continuous improvement." ;-)

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 Post subject: Re: Sterilizing Stems
PostPosted: Sun Jun 21, 2015 3:32 pm 
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Joined: Wed Nov 16, 2005 4:45 pm
Posts: 21
Location: Prague, Czech Republic
My simple protocol: I put stem into dish of 0.6 per cent sodium hypochlorite (with one drop of kitchen surfactant) for 1 1/2 to 2 hours. Then I place stem on sterile adsorbent paper, remove bracts with watchmaker forceps and cut the stem. Place peaces in tubes. Yield is better than 80 per cent. Adsorbent paper removes almost all cleach. Jaroslav


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